中国河北省卢龙地区猪札如病毒的基因多样性

Porcine sapoviruses (SaVs),which belong to the family Caliciviridae,have been considered potential zoonotic agents for human infection,and several cases have been reported in Asian countries. In this study,a total of 200 porcine fecal samples collected from Lulong county of China were tested. Among 200 samples,porcine sapoviruses were detected by RT-PCR in 17 samples (8.5%) showing their circulation in China. 14 out of 17 positive sapovirus strains were genetically related to the genogroup III (GIII) and we...     

Genetic Analysis of the Capsid Region of Human Astrovirus Serotype 3 Isolated in Japan

The total capsid region of astrovirus serotype 3 was analyzed using five isolates including four from Japan and one from the United Kingdom. The nucleic acid and deduced amino acid sequences in the region were homologous (over 97%). However, the sequences of serotype 3 were different from those of serotypes 1, 2, 4, 5, 6 and 8, especially in the C terminus.     

Detection and Genetic Characterization of Rabies Virus from Human Patients

Saliva and blood were collected from two patients who had not received post exposure prophylaxis in the cities of Wenzhou and Xinning respectively. Both patients were confirmed as positive for rabies by detection of rabies virus specific nucleoprotein antibodies in the sera by Western Blot. However, rabies virus specific RNA was only identified in the saliva collected from the patient in Wenzhou. Furthermore, the isolate Zhejiang Wz0 (H) was obtained by inoculating one-day-old suckling mice. Both nucleoprotein (N) and glycoprotein (G) genes from the isolate were amplified by RT-PCR and sequenced. Phylogenetic analysis indicated that the isolate belonged to classic rabies virus, and shared a higher homology with the street viruses from dogs in the main endemic areas in China and the street virus from dogs in Indonesia than with other known strains. Further comparison of the deduced amino acid sequences between the isolate and the vaccine strains used in China showed that the virus had a higher level of homology with the vaccine strain CTN than with the other vaccine strains (3aG, PV, PM and ERA). In particular, amino acid residues substitutions located in antigenic site Ⅲ in the G protein, which could react with the neutralizing antibodies, were observed. These results suggested that the virus belonged to the classic rabies virus, and both N and G genes diverged from the current vaccine strains used in China at either the nucleotide or the amino acid level.     

Assessment of Poliovirus Eradication in Japan: Genomic Analysis of Polioviruses Isolated from River Water and Sewage in Toyama Prefecture

Seventy-eight poliovirus strains isolated from river water and sewage in Toyama Prefecture, Japan, during 1993 to 1995 were characterized by the PCR-restriction fragment length polymorphism (RFLP) method and by partially sequencing the VP3 and VP1 regions of the viral genome. Of these isolates, 36 were identified as Sabin vaccine strains, and 42 were identified as vaccine variant strains that had less than 1.4% nucleotide divergence from the Sabin strains, including 7 isolates with patterns different from those of Sabin strains as determined by PCR-RFLP analysis. These findings suggest that wild-type poliovirus was not circulating in Toyama Prefecture.     

Genetic Analysis of Variation in Human Meiotic Recombination

The number of recombination events per meiosis varies extensively among individuals. This recombination phenotype differs between female and male, and also among individuals of each gender. In this study, we used high-density SNP genotypes of over 2,300 individuals and their offspring in two datasets to characterize recombination landscape and to map the genetic variants that contribute to variation in recombination phenotypes. We found six genetic loci that are associated with recombination phenotypes. Two of these (RNF212 and an inversion on chromosome 17q21.31) were previously reported in the Icelandic population, and this is the first replication in any other population. Of the four newly identified loci (KIAA1462, PDZK1, UGCG, NUB1), results from expression studies provide support for their roles in meiosis. Each of the variants that we identified explains only a small fraction of the individual variation in recombination. Notably, we found different sequence variants associated with female and male recombination phenotypes, suggesting that they are regulated by different genes. Characterization of genetic variants that influence natural variation in meiotic recombination will lead to a better understanding of normal meiotic events as well as of non-disjunction, the primary cause of pregnancy loss.     

Persistence of PCR-Detectable Bacteroides distasonis from Human Feces in River Water

To evaluate the persistence of PCR-detectable Bacteroides distasonis in surface water, whole human feces were dispersed into water from the Ohio River and incubated in flasks in the laboratory or in diffusion chambers in situ. Duplicate samples were taken daily, and material that pelleted at 16,000 × g was assayed by PCR. Persistence of PCR-detectable DNA from this anaerobe depended upon temperature and predation, two of the factors shown by others to influence the survival of aerobic bacteria detected by culture. B. distasonis was detected by PCR for at least 2 weeks at 4°C but for only 4 to 5 days at 14°C, 1 to 2 days at 24°C, and 1 day at 30°C. In filtered water or in the presence of cycloheximide, a eukaryotic inhibitor, persistence at 24°C was extended by at least a week.     

Continuous Presence of Noroviruses and Sapoviruses in Raw Sewage Reflects Infections among Inhabitants of Toyama,Japan (2006 to 2008)

Various genotypes of norovirus (NoV) (genogroup I genotype 1 [GI.1], -2, -4, -5, -8, -11, -12, and -14; GII.3, -4, -6, -7, -10, -13, -14, and -15), and sapovirus (SaV) (GI.1 and GI.2, GII.1, and GIV.1) were detected from raw sewage from April 2006 to March 2008, while limited numbers of genotypes of NoV (GI.8, GII.4, GII.6, and GII.13) and SaV (GII.3 and GIV.1) and of NoV (GII.4, GII.7, and GII.13) were detected from clinical cases and healthy children, respectively. During the winter 2006 to 2008, a large number of sporadic gastroenteritis outbreaks and many outbreaks caused by NoV GII.4 occurred among inhabitants in Toyama, Japan. The copy number of genomes of NoV GII detected from raw sewage changed in relation to the number of outbreaks. NoV strains of the same genotypes observed in both raw sewage and human specimens belonged to the same cluster by phylogenetic analysis and had almost identical nucleotide sequences among each genotype. These data suggest that NoVs and SaVs detected from raw sewage reflect the viruses circulating in the community, irrespective of symptoms, and that subclinical infections of NoV are common in Japan. Combined surveys of raw sewage with those of clinical cases help us to understand the relationship between infection of these viruses and gastroenteritis.Norovirus (NoV) and sapovirus (SaV), members of the Caliciviridae family, are considered to be a major cause of acute gastroenteritis in humans. Both NoV and SaV infect humans via the fecal-oral route and cause family or community-wide outbreaks, mainly in the winter season. NoVs are shed in feces at a level of 10⁵ to 10⁹ virus particles per gram during the symptomatic phase (32, 37), and viruses are continuously shed from patients after cessation of the symptoms (28, 37, 40). In addition, recent reports showed relatively high levels of shedding of the viruses from asymptomatic individuals (7, 8, 32, 37).NoVs and SaVs show high diversity in their genomes (5, 9). According to such a genetic diversity, they are classified into several genogroups (genogroup I [GI], GII, and GIV for human NoV and GI, GII, GIV, GV for human SaV) and further divided into many genotypes (NoV GI genotypes 1 to 14 [GI.1-14] and GII.1-17 and SaV GI.1-5, GII.1-6, GIV.1, and GV.1) (10, 17, 18). In 2006 to 2007, NoV GII.4 caused a large number of outbreaks of acute gastroenteritis worldwide (1, 11, 35, 43, 45). However, the other genotypes of NoV and SaV may infect humans asymptomatically and persist in the environment.Raw sewage could contain enteric viruses shed from affected people, and therefore, detectable viruses in raw sewage would reflect the actual state of the circulating viruses in the area. We previously reported that polioviruses in raw sewage and river water were isolated at the same time as oral vaccination in babies, and these isolates were derived from vaccine strains (13, 30). We also showed that the nucleotide sequences of echovirus type 13 isolated from river water were closely related to those from patients with aseptic meningitis during the outbreak in 2002 (14). For NoVs and SaVs, many epidemiological surveys have been conducted to determine the prevalence and virological properties of these viruses (42). Previous reports have shown that the nucleotide sequences of NoV strains from stools of outbreaks in nursing homes and from sewage were identical for an individual outbreak (26), and NoVs detected from gastroenteritis patients, domestic sewage, river water, and cultivated oysters in the area were related to each other (44). However, less is known about infection of the viruses with minor genotypes that are silently circulating in the population.In this study, we investigated NoVs and SaVs in raw sewage from 2006 to 2008 in Japan and compared the results with the viruses detected from clinical cases as well as healthy individuals to show the comprehensive prevalence of these viruses in the community.     

我国人类遗传资源采集活动现状分析与对策建议*

人类遗传资源是指含有人体基因组、基因等遗传物质的器官、组织、细胞等遗传材料及其产生的数据等资料。我国人类遗传资源极其丰富,合理利用人类遗传资源对推动我国生命科学、生物医药和临床研究具有重要意义。为有效保护、管理和利用我国人类遗传资源,管理部门严格依法依规开展人类遗传资源行政审批。通过梳理2021年我国人类遗传资源采集活动行政许可情况,结合工作实际,对存在问题进行分析,并提出对策建议。     

Detection of Peramivir and Laninamivir,New Anti-Influenza Drugs,in Sewage Effluent and River Waters in Japan

This is the first report of the detection of two new anti-influenza drugs, peramivir (PER) and laninamivir (LAN), in Japanese sewage effluent and river waters. Over about 1 year from October 2013 to July 2014, including the influenza prevalence season in January and February 2014, we monitored for five anti-influenza drugs—oseltamivir (OS), oseltamivir carboxylate (OC), zanamivir (ZAN), PER, and LAN—in river waters and in sewage effluent flowing into urban rivers of the Yodo River system in Japan. The dynamic profiles of these anti-influenza drugs were synchronized well with that of the numbers of influenza patients treated with the drugs. The highest levels in sewage effluents and river waters were, respectively, 82 and 41 ng/L (OS), 347 and 125 ng/L (OC), 110 and 35 ng/L (ZAN), 64 and 11 ng/L (PER), and 21 and 9 ng/L (LAN). However, application of ozone treatment before discharge from sewage treatment plants was effective in reducing the levels of these anti-influenza drugs in effluent. The effectiveness of the ozone treatment and the drug dependent difference in susceptibility against ozone were further evidenced by ozonation of a STP effluent in a batch reactor. These findings should help to promote further environmental risk assessment of the generation of drug-resistant influenza viruses in aquatic environments.     

Quantitative Detection of Human Adenoviruses in Wastewater and Combined Sewer Overflows Influencing a Michigan River

Enteric viruses are important pathogens found in contaminated surface waters and have previously been detected in waters of the Great Lakes. Human adenoviruses were monitored because of their high prevalence and persistence in aquatic environments. In this study, we quantified adenoviruses in wastewater, surface water, and combined sewer overflows (CSOs) by real-time PCR. Between August 2005 and August 2006, adenovirus concentrations in raw sewage, primary-treated effluent, secondary-treated effluent, and chlorinated effluent from a wastewater treatment plant in Michigan were examined. CSO samples (n = 6) were collected from a CSO retention basin in Grand Rapids, MI. Adenoviruses were detected in 100% of wastewater and CSO discharge samples. Average adenovirus DNA concentrations in sewage and CSOs were 1.15 × 10⁶ viruses/liter and 5.35 × 10⁵ viruses/liter, respectively. Adenovirus removal was <2 log₁₀ (99%) at the wastewater treatment plant. Adenovirus type 41 (60% of clones), type 12 (29%), type 40 (3%), type 2 (3%), and type 3 (3%) were isolated from raw sewage and primary effluents (n = 28). Six of 20 surface water samples from recreational parks at the lower Grand River showed virus concentrations above the real-time PCR detection limit (average, 7.8 × 10³ viruses/liter). This research demonstrates that wastewater effluents and wastewater-impacted surface waters in the lower Grand River in Michigan contain high levels of viruses and may not be suitable for full-body recreational activities. High concentrations of adenovirus in these waters may be due to inefficient removal during wastewater treatment and to the high persistence of these viruses in the environment.Enteric viruses are important waterborne pathogens. They are frequently isolated from feces-contaminated water and have been linked to numerous waterborne outbreaks (9, 34, 42, 61). This group of pathogens includes adenoviruses, enteroviruses, hepatitis A virus, noroviruses, and rotavirus. In the Great Lakes region, enteric viruses were isolated from recreational beaches and groundwater for municipal usage, indicating an elevated public health risk in consuming or coming into contact with these waters (15, 69). Although recent developments in molecular detection assays substantially increase the detection of viruses from waters, from a management standpoint it is impractical to test all viruses when determining the microbial quality of water. Here we propose that adenovirus monitoring can be used to examine wastewater impacts on surface water quality.Adenoviruses, which have a high prevalence in water, have been suggested as preferred candidates as index organisms for viral pathogens because they fit most criteria for an ideal indicator (19, 33, 38, 54). It is estimated that more than 90% of the human population is seropositive for one or more serotypes of adenoviruses (11, 68). Human adenoviruses (HAdVs) are present at a higher frequency in sewage than are other enteric viruses (54) and are excreted in high concentrations from infected patients (up to 10¹¹ viral particles per gram of feces) (68).Adenoviruses were first isolated from humans and identified as the causative agent of epidemic febrile respiratory disease among military recruits in the 1950s (30, 55). Human adenoviruses are the second most important viral pathogen of infantile gastroenteritis, after rotavirus (3, 10, 44, 51, 58, 62, 65). Serotypes of adenoviruses have been found to cause symptomatic infections in several organ systems, including the respiratory system (pharyngitis, acute respiratory disease, and pneumonia), eye (conjunctivitis), gastrointestinal tract (gastroenteritis), central nervous system (meningoencephalitis), and genitalia (urethritis and cervicitis) (8, 37). Human adenovirus types 40 and 41 have been associated with gastroenteritis in children, while human adenovirus type 4 is linked to persistent epidemics of acute respiratory disease in the United States (10, 49). It was estimated that 2 to 7% of all lower respiratory tract illnesses in children may be caused by adenoviruses (5, 17).Transmission routes of adenoviruses include the fecal-oral route and inhalation of aerosols. Adenoviruses have been associated with outbreaks in different settings, including military camps (7, 36, 40), hospitals (6, 28, 32), day care centers (1, 38), and schools (27). Waterborne outbreaks due to adenoviruses have also involved swimming pools (53, 64).It is hypothesized that combined sewer overflows (CSOs; where storm water and untreated sewage are combined) may contribute high concentrations of waterborne pathogens, especially viruses, which in turn may pose an adverse risk to human health. In older cities of Michigan, such as Detroit, East Lansing, and Grand Rapids, major contributors to microbial contamination of surface water during high-rainfall events include discharges from sanitary sewer systems and combined sewer systems. The federal government''s effort to control CSOs started in 1994, when the U.S. EPA published the CSO Control Policy as the national framework. In Michigan, the first CSO policy was drafted by the Department of Environmental Quality in 1983. However, the first noncontested permit requiring a long-term CSO correction program was issued to the Grand Rapids wastewater treatment plant (WWTP) only in Fall 1988, following a large CSO event in the city that affected water quality downstream, in Grand Haven (50). To date, Michigan communities have eliminated 75% of the 613 untreated CSO outfalls that existed in the year 1988, and the remaining 25% are scheduled for correction/elimination through implementation of long-term control plans. However, water quality after CSO or any sewage spill remains a public health concern to individuals via recreational water exposure at recreational parks and beaches downstream of discharge sites.The aim of this study was to evaluate the presence and concentration of adenoviruses in sewage and in the Grand River in the state of Michigan. Raw sewage, wastewater effluent, CSO discharges, and surface water in the lower Grand River were surveyed for the occurrence and concentration of human adenoviruses. Real-time PCR was used for quantification. Predominant adenovirus genotypes in sewage were determined, and the efficiency of virus removal during wastewater treatment was evaluated.     

Analysis of Dengue Virus Genetic Diversity during Human and Mosquito Infection Reveals Genetic Constraints

Dengue viruses (DENV) cause debilitating and potentially life-threatening acute disease throughout the tropical world. While drug development efforts are underway, there are concerns that resistant strains will emerge rapidly. Indeed, antiviral drugs that target even conserved regions in other RNA viruses lose efficacy over time as the virus mutates. Here, we sought to determine if there are regions in the DENV genome that are not only evolutionarily conserved but genetically constrained in their ability to mutate and could hence serve as better antiviral targets. High-throughput sequencing of DENV-1 genome directly from twelve, paired dengue patients’ sera and then passaging these sera into the two primary mosquito vectors showed consistent and distinct sequence changes during infection. In particular, two residues in the NS5 protein coding sequence appear to be specifically acquired during infection in Ae. aegypti but not Ae. albopictus. Importantly, we identified a region within the NS3 protein coding sequence that is refractory to mutation during human and mosquito infection. Collectively, these findings provide fresh insights into antiviral targets and could serve as an approach to defining evolutionarily constrained regions for therapeutic targeting in other RNA viruses.     

Human genome efforts in Japan

Some project-oriented biosciences have been promoted cooperatively by three governmental agencies in Japan: the Ministry of Education, Science and Culture, the Ministry of Health and Welfare, and the Science and Technology Agency. All three agencies have increased their budgetary requests for human genome analyses for FY 1991. The Panel on Life Sciences (chaired by Dr. W. Mori, former president of the University of Tokyo) of the Science and Technology Council of Japan which is chaired by the Prime Minister has decided to organize a working group to suggest how best to coordinate efforts in human genome analyses in Japan. The genome project was initially promoted by the Science and Technology Agency through a program for the design and construction of automated machines for DNA sequencing. Work is ongoing at the Institute of Physical and Chemical Research to integrate, by system engineering, instruments that can separate DNA fragments, perform plaque selection, carry out dideoxy reaction, and read the resulting DNA sequence. However, scientists now realize the enormity of the tasks of compiling DNA sequence data and of mapping the genes and fragments obtained, and efforts are being made to solve these problems. Academic societies have organized symposia to promote general interest in this subject. The most important way for Japan to contribute to research on human genome analyses, however, may be in the evaluation of supporting mechanisms (technical assistance and research resources) and in the recognition and preparation of the transition of biology to a big science approach.     

Identification of Bacterial DNA Markers for the Detection of Human Fecal Pollution in Water

We used genome fragment enrichment and bioinformatics to identify several microbial DNA sequences with high potential for use as markers in PCR assays for detection of human fecal contamination in water. Following competitive solution-phase hybridization of total DNA from human and pig fecal samples, 351 plasmid clones were sequenced and were determined to define 289 different genomic DNA regions. These putative human-specific fecal bacterial DNA sequences were then analyzed by dot blot hybridization, which confirmed that 98% were present in the source human fecal microbial community and absent from the original pig fecal DNA extract. Comparative sequence analyses of these sequences suggested that a large number (43.5%) were predicted to encode bacterial secreted or surface-associated proteins. Deoxyoligonucleotide primers capable of annealing to a subset of 26 of the candidate sequences predicted to encode factors involved in interactions with host cells were then used in the PCR and did not amplify markers in DNA from any additional pig fecal specimens. These 26 PCR assays exhibited a range of specificity in tests with 11 other animal sources, with more than half amplifying markers only in specimens from dogs or cats. Four assays were more specific, detecting markers only in specimens from humans, including those from 18 different human populations examined. We then demonstrated the potential utility of these assays by using them to detect human fecal contamination in several impacted watersheds.     

金沙江河谷地区岩羊种群遗传多样性分析

岩羊为国家Ⅱ级保护动物。目前,由于人类活动范围日益扩大,其栖息地遭到了不同程度的破坏,岩羊种群数量和分布范围呈明显下降趋势。为了及时制定科学的保护策略必须了解它们的种群遗传结构。本文以粪便为研究材料,对来自金沙江河谷地区林线以上岩羊和林线以下矮个子岩羊共4个地理种群的169份有效样品进行线粒体CR全序列分析,共检测出210个变异位点,定义了4种单倍型,单倍型多样性为0.68,核苷酸多样性为0.0242,显示种群整体遗传多样性水平较低。基于最大简约法构建的系统发育树中,金沙江河谷地区的4个地理种群被划分到四川种群的2个亚分支中,但云南曲宗贡的部分岩羊和四川竹巴笼的矮个子岩羊单倍型存在共享现象。根据分子钟计算,林线上下岩羊分化时间在39~32万年前,在中更新世早中期(105~36万年前)气候影响下,导致岩羊在金沙江河谷高低海拔之间相互迁移。对于岩羊在历史上所表现出的潜在迁徙能力,我们建议将白马雪山自然保护区到竹巴笼自然保护区之间的金沙江河谷地区作为岩羊和矮个子岩羊的栖息地整体保护。     

Hollow-Fiber Ultrafiltration and PCR Detection of Human-Associated Genetic Markers from Various Types of Surface Water in Florida

Hollow-fiber ultrafiltration (HFUF) and PCR were combined to detect human-associated microbial source tracking marker genes in large volumes of fresh and estuarine Florida water. HFUF allowed marker detection when membrane filtration did not, demonstrating HFUF''s ability to facilitate detection of diluted targets by PCR in a variety of water types.Microbial source tracking (MST) uses analytical methods to determine the possible source(s) of fecal contamination in environmental waters (reviewed in reference 14). Human fecal pollution (i.e., sewage) is known to contain pathogens that pose serious health risks (6, 8). Identifying sewage impacts can prevent illness, through public warnings and environmental remediation. Detection of sewage has been demonstrated with genetic markers from microorganisms associated with humans (3, 4, 7, 11, 15), each of which has pros and cons (5, 14). A useful addition to MST would be the concentration of samples when the standard protocol (e.g., filtering 500 ml) (5) is not feasible due to turbidity of the water or dilution of the target(s). Hollow-fiber ultrafiltration (HFUF) concentrates particulates from water, with little increase in total assay time (9), while simultaneously retaining parasites, bacteria, and viruses (13). Dead-end HFUF of culturable enterococci in recreational water was successfully demonstrated in Florida (9) and California (10). The objective of this study was to assess the utility of HFUF for concentration of higher-volume samples that would permit detection of diluted MST markers from surface water samples by PCR. Three markers representing a variety of microbial types were targeted: (i) human-associated Bacteroidales (Bacteria), (ii) human polyomaviruses (HPyVs; virus), and (iii) Methanobrevibacter smithii (Archaea).Water samples were collected in Hillsborough Country, FL. Freshwater originated from the unimpacted headwaters of the Hillsborough River (28°5′56.98"N, 82°18′44.05"W) and estuarine water from remediated Ben T. Davis Beach (27°58′5.71"N, 82°34′26.46"W). Sewage-impacted water samples were collected from Sweetwater Creek (27°59′57.27"N, 82°33′37.10"W), which received approximately 500,000 gallons of sewage from a sewer main break. Samples were collected within 5 days and 45 days of the spill. The 5-day sample was collected after the spill was reported, while the 45-day sample was collected to test a more diluted sample of the same water. Raw sewage (influent) for spiked samples was collected on the day of the HFUF experiment from a Hillsborough County wastewater treatment plant that processes 9 million gallons daily.Initial HFUF samples consisted of Dulbecco''s phosphate-buffered saline (D-PBS; pH 7.4) spiked with 5 ml of raw sewage (5 × 10⁻⁴ dilution). Spiked environmental samples included 10 liters of ambient water spiked with 5 or 2.5 ml of raw sewage. Fecal indicator bacterium (FIB) concentrations of the unamended samples were determined by membrane filtration using standard methods for Enterococcus spp., Escherichia coli (16), and fecal coliforms (2) to demonstrate the ambient microbial water quality at the sites. Water samples were first tested for existing MST markers before spiking with sewage. Ten-liter samples of unamended ambient water (Ben T. Davis Beach and Sweetwater Creek) were collected to assess the method''s ability to detect diluted targets following method verification with spiked experiments. Subsequent sewage spikes into unamended samples were performed to ensure that any lack of detection using the HFUF protocol was not due to inhibition. Water samples were collected for processing by the standard protocols from each site in concert with HFUF samples (5). The Sweetwater Creek sample allowed only 300 ml to be filtered instead of the standard 500 ml due to its high turbidity (160 nephelometric turbidity units [NTU]).The design and operation of the recreational dead-end concentrator (Rec DEC) have been previously described (9, 10). A new F80A Hemoflow polysulfone high-flux capillary dialyzer (Fresenius Medical Care North America, Lexington, MA) was used for each sample. Samples were fed into the filters by a peristaltic pump in approximately 6.5 min. All Rec DEC components were cleaned between sample feedings with 10% bleach followed by 10% sodium thiosulfate. Elution of each filter was performed using 250 ml of 4 M urea-50 mM lysine (pH 9.0) that was incubated in the filter fibers for 2 min before collection of the retentate.The pH of each retentate was adjusted to 3.5 to retain HPyVs (11, 12), and then 25 ml was filtered through a 0.45-μm-pore-size nitrocellulose membrane. Each membrane was placed into a Mo Bio PowerSoil microcentrifuge tube (Mo Bio Laboratories, Carlsbad, CA), and extraction of total DNA was performed as previously described (5). Conventional PCR was used to determine the presence or absence of MST markers. Assays targeted a species-specific region of the 16S rRNA gene of human-associated Bacteroidales (3) with slight modifications (5), the conserved T antigen of human BK and JC polyomaviruses (1, 5), or the nifH gene of Methanobrevibacter smithii (5, 15).Each marker was detected in all HFUF spiked samples (Table ​(Table1).1). The procedure was also successful in detecting the markers with the reduced sewage spike volume (2.5 ml) for the Ben T. Davis Beach and Sweetwater Creek samples (Table ​(Table1).1). Detection of smaller amounts of spiked sewage was not attempted in this study; however, previous work determined detection limits of 10⁻³ to 10⁻⁴ dilution for HPyVs and M. smithii (5) and 10⁻⁵ to 10⁻⁶ dilution for Bacteroidales (3, 5).

TABLE 1.

PCR detection of human-associated MST markers from surface water samples following HFUF with Rec DEC

Genetic Analysis of Natural Populations of DROSOPHILA MELANOGASTER in Japan. III. Genetic Variability of Inducing Factors of Amylase and Fitness

"Inducibility" of amylase in Drosophila melanogaster was defined and investigated in a natural population from Japan. Inducibility represents the effects of factors remote from the structural gene that control the amount of enzyme produced. Inducibility of an isogenic line is measured as the ratio of the enzyme's specific activity in two different inducing environments. There was considerable genetic variability with respect to inducibility of amylase in 44 isogenic lines derived from a natural population of D. melanogaster . Net fitness and its components in these isogenic lines were also measured. The results indicated that, although the inducibility of the enzyme was positively correlated with the net fitness (r_g = 0.63 ± 0.2), the enzyme activities in the normal medium were not (r_g = 0.12 ± 0.37). The analysis of the data shows that the differences in inducing factors are mainly responsible for the differences in the fitness of lines and are the genetic materials for the adaptive evolution of organisms.     

Genetic variations ofCottus nozawae populations from five tributaries of the Shiribetsu River of southern Hokkaido,Japan

To estimate genetic differentiation and heterogeneity in the landlocked river sculpin,Cottus nozawae, between tributary populations in the same river-system, 107 specimens were captured from 5 tributaries of the Shiribetsu river (course length 128 km), Hokkaido Island and surveyed for allozyme variations and restriction fragment length polymorphisms of mitochondrial DNA (mtDNA). Three and two alleles were seen at theIdh-2 andPgm loci, respectively, but only one locus,Idh-2, out of twenty loci examined was regarded as polymorphic, since the frequency of the most common allele did not exceed 0.95. Three different mtDNA haplotypes were detected, there being replacement of them between the tributary populations. Heterogeneities of allele and haplotype frequencies were significant between some tributary populations, suggesting that genetic differentiation has occurred between them.